Jacob Schaefer

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Name: Schaefer, Jacob

Organization: Washington University , USA

Department: Department of Chemistry

Title: Professor(PhD)

TOPICS

Material Chemistry

Chemicals
References

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis

Co-reporter:Hao Yang, Manmilan Singh, Sung Joon Kim, Jacob Schaefer

Biochimica et Biophysica Acta (BBA) - Biomembranes 2017 Volume 1859, Issue 11(Issue 11) pp:

Publication Date(Web):1 November 2017

DOI:10.1016/j.bbamem.2017.08.003

•E. faecalis cell-wall tertiary structure has been characterized by solid-state NMR.•d-Alanine in wall teichoic acids is 18% of that in peptidoglycan.•Peptidoglycan bridge-linking is 85%, and cross-linking is 48%.•In peptidoglycan, 40% of stems are tripeptide, and 12% are tetra/pentapeptide.•E. faecalis peptidoglycan has a hybrid short-bridge architecture.Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13C and 15N specific labels in d- and l-alanine and l-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l-alanyl carbonyl‑carbon bridge labels to d-[3-13C]alanyl and l-[ε-15N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.Download high-res image (257KB)Download full-size image

Dual Mode of Action for Plusbacin A3 in Staphylococcus aureus

Co-reporter:Robert D. O’Connor, Manmilan Singh, James Chang, Sung Joon KimMichael VanNieuwenhze, Jacob Schaefer

The Journal of Physical Chemistry B 2017 Volume 121(Issue 7) pp:

Publication Date(Web):January 30, 2017

DOI:10.1021/acs.jpcb.6b11039

We have used C{F}, N{F}, and N{P} rotational-echo double resonance NMR to determine the location and conformation of 19F and 15N double-labeled plusbacin A3 and of double-labeled deslipo-plusbacin A3, each bound to the cell walls of whole cells of Staphyloccocus aureus grown in media containing [1-13C]glycine. The 31P is primarily in wall teichoic acid. Approximately 25% of plusbacin headgroups (the cyclic depsipeptide backbone) are in a closed conformation (N–F separation of 6 Å), while 75% are in a more open conformation (N–F separation of 12 Å). The closed headgroups have no contact with wall teichoic acid, whereas the open headgroups have a strong contact. This places the closed headgroups in hydrophobic regions of the cell wall and the open headgroups in hydrophilic regions. None of the plusbacin tails have contact with the 31P of either wall teichoic acid or the cell membrane and thus are in hydrophobic regions of the cell wall. In addition, both heads and tails of plusbacin A3 have contact with the glycyl 13C incorporated in cell-wall peptidoglycan pentaglycyl bridges and with 13C-labeled purines near the membrane surface. We interpret these results in terms of a dual mode of action for plusbacin A3: first, disruption of the peptidoglycan layer nearest to the membrane surface by closed-conformation plusbacin A3 leading to an inhibition of chain extension by transglycosylation; second, thinning and disruption of the membrane (possibly including disruption of ATP-binding cassette transporters embedded in the membrane) by open-conformation plusbacin A3, thereby leading to release of ATP to the hydrophilic regions of the cell wall and subsequent binding by plusbacin A3.

Cross-Link Formation and Peptidoglycan Lattice Assembly in the FemA Mutant of Staphylococcus aureus

Co-reporter:Sung Joon Kim, Manmilan Singh, Shasad Sharif, and Jacob Schaefer

Biochemistry 2014 Volume 53(Issue 9) pp:

Publication Date(Web):February 11, 2014

DOI:10.1021/bi4016742

Staphylococcus aureus FemA mutant grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-13C]alanine, l-[3-13C]alanine, [2-13C]glycine, and l-[5-19F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between 13C-labeled alanines and 19F-labeled lysine incorporated in the peptidoglycan. The alanyl 13C labels were preselected for REDOR measurement by their proximity to the glycine label using 13C–13C spin diffusion. The observed 13C–13C and 13C–19F distances are consistent with a tightly packed, hybrid architecture containing both parallel and perpendicular stems in a repeating structural motif within the peptidoglycan.

The Isotridecanyl Side Chain of Plusbacin-A3 Is Essential for the Transglycosylase Inhibition of Peptidoglycan Biosynthesis

Co-reporter:Sung Joon Kim, Manmilan Singh, Aaron Wohlrab, Tsyr-Yan Yu, Gary J. Patti, Robert D. O’Connor, Michael VanNieuwenhze, and Jacob Schaefer

Biochemistry 2013 Volume 52(Issue 11) pp:

Publication Date(Web):February 19, 2013

DOI:10.1021/bi4000222

Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm, and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer, thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [2H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [2H]pb-A3 has an isotopically 2H-labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [2H]pb-A3 bound to [1-13C]glycine-labeled whole cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [2H]pb-A3 during the growth of S. aureus resulted in the accumulation of Park’s nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.

Locations of the Hydrophobic Side Chains of Lipoglycopeptides Bound to the Peptidoglycan of Staphylococcus aureus

Co-reporter:Sung Joon Kim, Kelly S. E. Tanaka, Evelyne Dietrich, Adel Rafai Far, and Jacob Schaefer

Biochemistry 2013 Volume 52(Issue 20) pp:

Publication Date(Web):April 22, 2013

DOI:10.1021/bi400054p

Glycopeptides whose aminosugars have been modified by attachment of hydrophobic side chains are frequently active against vancomycin-resistant microorganisms. We have compared the conformations of six such fluorinated glycopeptides (with side chains of varying length) complexed to cell walls labeled with d-[1-13C]alanine, [1-13C]glycine, and l-[ε-15N]lysine in whole cells of Staphylococcus aureus. The internuclear distances from 19F of the bound drug to the 13C and 15N labels of the peptidoglycan, and to the natural abundance 31P of lipid membranes and teichoic acids, were determined by rotational-echo double resonance NMR. The drugs did not dimerize, and their side chains did not form membrane anchors but instead became essential parts of secondary binding to pentaglycyl bridge segments of the cell-wall peptidoglycan.

Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy

Co-reporter:Sung Joon Kim, Manmilan Singh, Maria Preobrazhenskaya, and Jacob Schaefer

Biochemistry 2013 Volume 52(Issue 21) pp:

Publication Date(Web):April 25, 2013

DOI:10.1021/bi4005039

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-13C]alanine and l-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C–15N average distance of 4.1–4.4 Å between d- and l-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.

Chain Packing in Glassy Polymers by Natural-Abundance 13C−13C Spin Diffusion Using 2D Centerband-Only Detection of Exchange

Co-reporter:Manmilan Singh

Journal of the American Chemical Society 2011 Volume 133(Issue 8) pp:2626-2631

Publication Date(Web):February 9, 2011

DOI:10.1021/ja109104f

The proximities of specific subgroups of nearest-neighbor chains in glassy polymers are revealed by distance-dependent 13C−13C dipolar couplings and spin diffusion. The meassurement of such proximities is practical even with natural-abundance levels of 13C using a 2D version of centerband-only detection of exchange (CODEX). Two-dimensional CODEX is a relaxation-compensated experiment that avoids the problems associated with variations in T1(C)’s due to dynamic site heterogeneity in the glass. Isotropic chemical shifts are encoded in the t1 preparation times before and after mixing, and variations in T2’s are compensated by an S0 reference (no mixing). Data acquisition involves acquisition of an S0 reference signal on alternate scans, and the active control of power amplifiers, to achieve stability and accuracy over long accumulation times. The model system to calibrate spin diffusion is the polymer itself. For a mixing time of 200 ms, only 13C−13C pairs separated by one or two bonds (2.5 Å) show cross peaks, which therefore identify reference intrachain proximities. For a mixing time of 1200 ms, 5 Å interchain proximities appear. The resulting cross peaks are used in a simple and direct way to compare nonrandom chain packing for two commercial polycarbonates with decidedly different mechanical properties.

“Development of REDOR rotational-echo double-resonance NMR” by Terry Gullion and Jacob Schaefer [J. Magn. Reson. 81 (1989) 196–200]

Co-reporter:Jacob Schaefer

Journal of Magnetic Resonance 2011 213(2) pp: 421-422

Publication Date(Web):

DOI:10.1016/j.jmr.2011.08.012

Variability in C3-Plant Cell-Wall Biosynthesis in a High-CO2 Atmosphere by Solid-State NMR Spectroscopy

Co-reporter:Tsyr-Yan Yu ; Manmilan Singh ; Shigeru Matsuoka ; Gary J. Patti ; Gregory S. Potter

Journal of the American Chemical Society 2010 Volume 132(Issue 18) pp:6335-6341

Publication Date(Web):April 15, 2010

DOI:10.1021/ja909796y

We have used a frequency-selective rotational-echo double-resonance (REDOR) solid-state NMR experiment to measure the concentrations of glycine−glycine pairs in proteins (and protein precursors) of intact leaves of plants exposed to both high- and low-CO2 atomospheres. The results are interpreted in terms of differences in cell-wall biosynthesis between plant species. We illustrate this variability by comparing the assimilation of label in cheatgrass and soybean leaves labeled using 15N-fertilizer and 13CO2 atmospheres. Cheatgrass and soybean are both C3 plants but differ in their response to a high-CO2 environment. Based on REDOR results, we determined that cheatgrass (a plant that seems likely to flourish in future low-water, high-CO2 environments) routes 2% of the assimilated carbon label that remains in the leaf after 1 h in a 600-ppm 13CO2 atmosphere to glycine-rich protein (or its precursors), a structural component of cell walls cross-linked to lignins. In contrast, soybean under the same conditions routes none of its assimilated carbon to glycine-rich protein.

Oxygen-17 Appears Only in Protein in Water-Stressed Soybean Leaves Labeled by 17O2

Co-reporter:Terry Gullion ; Tsyr-Yan Yu ; Manmilan Singh ; Gary J. Patti ; Gregory S. Potter

Journal of the American Chemical Society 2010 Volume 132(Issue 31) pp:10802-10807

Publication Date(Web):July 19, 2010

DOI:10.1021/ja102264w

We have used a rotational-echo adiabatic-passage double-resonance 13C{17O} solid-state NMR experiment to prove that the glycine produced in the oxygenase reaction of ribulose bisphosphate carboxylase-oxygenase is incorporated exclusively into protein (or protein precursors) of intact, water-stressed soybean leaves exposed to 13CO2 and 17O2. The water stress increased stomatal resistance and decreased gas exchange so that the Calvin cycle in the leaf chloroplasts was no more than 35% 13C isotopically enriched. Labeled O2 levels were sufficient, however, to increase the 17O isotopic concentration of oxygenase products 20-fold over the natural-abundance level of 0.04%. The observed direct incorporation of glycine into protein shows that water stress suppresses photorespiration in soybean leaves.

Solid-State NMR Investigations of the Unusual Effects Resulting from the Nanoconfinement of Water within Amphiphilic Crosslinked Polymer Networks

Co-reporter:Ryutaro Ohashi;Jeremy W. Bartels;Jinqi Xu;Karen L. Wooley

Advanced Functional Materials 2009 Volume 19( Issue 21) pp:3404-3410

Publication Date(Web):

DOI:10.1002/adfm.200900846

Abstract

Two types of solid-state ¹⁹F NMR spectroscopy experiments are used to characterize phase-separated hyperbranched fluoropolymer–poly(ethylene glycol) (HBFP–PEG) crosslinked networks. Mobile (soft) domains are detected in the HBFP phase by a rotor-synchronized Hahn echo under magic-angle spinning conditions, and rigid (hard) domains by a solid echo with no magic-angle spinning. The mobility of chains is detected in the PEG phase by ¹H ¹³C cross-polarization transfers with ¹H spin-lock filters with and without magic-angle spinning. The interface between HBFP and PEG phases is detected by a third experiment, which utilized a ¹⁹F ¹H–(spin diffusion)–¹H ¹³C double transfer with ¹³C solid-echo detection. The results of these experiments show that composition-dependent PEG inclusions in the HBFP glass rigidify on hydration, consistent with an increase in macroscopic tensile strength.

Characterization of Peptidoglycan in Fem-Deletion Mutants of Methicillin-Resistant Staphylococcus aureus by Solid-State NMR

Co-reporter:Shasad Sharif, Sung Joon Kim, Harald Labischinski and Jacob Schaefer

Biochemistry 2009 Volume 48(Issue 14) pp:

Publication Date(Web):March 23, 2009

DOI:10.1021/bi801750u

Compositional analysis of the peptidoglycan (PG) of a wild-type methicillin-resistant Staphylococcus aureus and its fem-deletion mutants has been performed on whole cells and cell walls using stable-isotope labeling and rotational-echo double-resonance NMR. The labels included [1-13C,15N]glycine and l-[ε-15N]lysine (for a direct measure of the number of glycyl residues in the bridging segment), [1-13C]glycine and l-[ε-15N]lysine (concentration of bridge links), and d-[1-13C]alanine and [15N]glycine (concentrations of cross-links and wall teichoic acids). The bridging segment length changed from 5.0 glycyl residues (wild-type strain) to 2.5 ± 0.1 (FemB) with modest changes in cross-link and bridge-link concentrations. This accurate in situ measurement for the FemB mutant indicates a heterogeneous PG structure with 25% monoglycyl and 75% triglycyl bridges. When the bridging segment was reduced to a single glycyl residue 1.0 ± 0.1 (FemA), the level of cross-linking decreased by more than 20%, resulting in a high concentration of open N-terminal glycyl segments.

Characterization of the Peptidoglycan of Vancomycin-Susceptible Enterococcus faecium

Co-reporter:Gary J. Patti, Sung Joon Kim and Jacob Schaefer

Biochemistry 2008 Volume 47(Issue 32) pp:

Publication Date(Web):July 19, 2008

DOI:10.1021/bi8008032

Vancomycin and other antibacterial glycopeptide analogues target the cell wall and affect the enzymatic processes involved with cell-wall biosynthesis. Understanding the structure and organization of the peptidoglycan is the first step in establishing the mode of action of these glycopeptides. We have used solid-state NMR to determine the relative concentrations of stem-links (64%), bridge-links (61%), and cross-links (49%) in the cell walls of vancomycin-susceptible Enterococcus faecium (ATTC 49624). Furthermore, we have determined that in vivo only 7% of the peptidoglycan stems terminate in d-Ala-d-Ala, the well-known vancomycin-binding site. Presumably, d-Ala-d-Ala is cleaved from uncross-linked stems in mature peptidoglycan by an active carboxypeptidase. We believe that most of the few pentapeptide stems ending in d-Ala-d-Ala occur in the template and nascent peptidoglycan strands that are crucial for cell-wall biosynthesis.

Vancomycin Derivative with Damaged d-Ala-d-Ala Binding Cleft Binds to Cross-linked Peptidoglycan in the Cell Wall of Staphylococcus aureus

Co-reporter:Sung Joon Kim, Shigeru Matsuoka, Gary J. Patti and Jacob Schaefer

Biochemistry 2008 Volume 47(Issue 12) pp:

Publication Date(Web):February 27, 2008

DOI:10.1021/bi702232a

Des-N-methylleucyl-4-(4-fluorophenyl)benzyl-vancomycin (DFPBV) retains activity against vancomycin-resistant pathogens despite its damaged d-Ala-d-Ala binding cleft. Using solid-state nuclear magnetic resonance (NMR), a DFPBV binding site in the cell walls of whole cells of Staphylococcus aureus has been identified. The cell walls were labeled with d-[1-13C]alanine, [1-13C]glycine, and l-[ϵ-15N]lysine. Internuclear distances from 19F of the DFPBV to the 13C and 15N labels of the cell-wall peptidoglycan were determined by rotational-echo double-resonance (REDOR) NMR. The 13C{19F} and 15N{19F} REDOR spectra show that, in situ, DFPBV binds to the peptidoglycan as a monomer with its vancosamine hydrophobic side chain positioned near a pentaglycyl bridge. This result suggests that the antimicrobial activity of other vancosamine-modified glycopeptides depends upon both d-Ala-d-Ala stem-terminus recognition (primary binding site) and stem-bridge recognition (secondary binding site).

15N{31P} REDOR NMR Studies of the Binding of Phosphonate Reaction Intermediate Analogues to Saccharomyces cerevisiae Lumazine Synthase†

Co-reporter:Tsyr-Yan Yu, Robert D. O’Connor, Astrid C. Sivertsen, Colby Chiauzzi, Barbara Poliks, Markus Fischer, Adelbert Bacher, Ilka Haase, Mark Cushman and Jacob Schaefer

Biochemistry 2008 Volume 47(Issue 52) pp:13942-13951

Publication Date(Web):December 3, 2008

DOI:10.1021/bi8015789

Lumazine synthase catalyzes the reaction of 5-amino-6-d-ribitylamino-2,4(1H,3H)-pyrimidinedione (1) with (S)-3,4-dihydroxybutanone 4-phosphate (2) to afford 6,7-dimethyl-8-d-ribityllumazine (3), the immediate biosynthetic precursor of riboflavin. The overall reaction implies a series of intermediates that are incompletely understood. The 15N{31P} REDOR NMR spectra of three metabolically stable phosphonate reaction intermediate analogues complexed to Saccharomyces cerevisiae lumazine synthase have been obtained at 7 and 12 T. Distances from the phosphorus atoms of the ligands to the side chain nitrogens of Lys92, His97, Arg136, and His148 have been determined. These distances were used in combination with the X-ray crystal coordinates of one of the intermediate analogues complexed with the enzyme in a series of distance-restrained molecular dynamics simulations. The resulting models indicate mobility of the Lys92 side chain, which could facilitate the exchange of inorganic phosphate eliminated from the substrate in one reaction, with the organic phosphate-containing substrate necessary for the next reaction.

Hydrophobic Side-Chain Length Determines Activity and Conformational Heterogeneity of a Vancomycin Derivative Bound to the Cell Wall of Staphylococcus aureus

Co-reporter:Sung Joon Kim and Jacob Schaefer

Biochemistry 2008 Volume 47(Issue 38) pp:

Publication Date(Web):August 30, 2008

DOI:10.1021/bi800838c

Disaccharide-modified glycopeptides with hydrophobic side chains are active against vancomycin-resistant enterococci and vancomycin-resistant Staphylococcus aureus. The activity depends on the length of the side chain. The benzyl side chain of N-(4-fluorobenzyl)vancomycin (FBV) has the minimal length sufficient for enhancement in activity against vancomycin-resistant pathogens. The conformation of FBV bound to the peptidoglycan in whole cells of S. aureus has been determined using rotational-echo double resonance NMR by measuring internuclear distances from the 19F of FBV to 13C and 15N labels incorporated into the cell-wall peptidoglycan. The hydrophobic side chain and aglycon of FBV form a cleft around the pentaglycyl bridge. FBV binds heterogeneously to the peptidoglycan as a monomer with the 19F positioned near the middle of the pentaglycyl bridge, approximately 7 Å from the bridge link. This differs from the situation for N-(4-(4-fluorophenyl)benzyl)vancomycin complexed to the peptidoglycan where the 19F is located at the end of pentaglycyl bridge, 7 Å from the cross-link.

Slow motion in [ring-fluoro]polycarbonate by CODEX

Co-reporter:Thomas K. Weldeghiorghis;Dirk Stueber

Journal of Polymer Science Part B: Polymer Physics 2008 Volume 46( Issue 11) pp:1062-1066

Publication Date(Web):

DOI:10.1002/polb.21439

Abstract

Chain dynamics in [ring-fluoro]polycarbonate (an A-B alternating copolymer that has a single fluorine substituent on every fourth main chain ring) have been characterized by centerband only detection of exchange (CODEX) and rotating-frame ¹³C spin-lattice relaxation. The slow motions detected by CODEX are facilitated by a mechanically active lattice reorganization that permits a flip of the fluorinated ring about its C₂ axis. Nonfluorinated rings undergo small-amplitude reorientations and C₂ flips, both of which are fast and not CODEX active. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1062–1066, 2008

Dipolar double-quantum filtered rotational-echo double resonance

Co-reporter:Shigeru Matsuoka

Magnetic Resonance in Chemistry 2007 Volume 45(Issue S1) pp:S61-S64

Publication Date(Web):21 DEC 2007

DOI:10.1002/mrc.2076

The homonuclear dipolar coupling of a directly bonded ¹³C¹³C pair has been used to create a dipolar double-quantum filter (D-DQF) to remove the natural-abundance ¹³C background in ¹³C{²H} rotational-echo double-resonance (REDOR) experiments. The most efficient version of this experiment has the D-DQF excitation and reconversion preceding the REDOR evolution period. Calculated and observed ¹³C{²H}D-DQF-REDOR dephasings were in agreement for a test sample of mixed recrystallized labeled alanines. Copyright © 2007 John Wiley & Sons, Ltd.

Molecular motion of polycarbonate included in γ-cyclodextrin

Co-reporter:Younkee Paik;Barbara Poliks;Cristian C. Rusa;Alan E. Tonelli

Journal of Polymer Science Part B: Polymer Physics 2007 Volume 45(Issue 11) pp:1271-1282

Publication Date(Web):16 APR 2007

DOI:10.1002/polb.21112

Molecular motions of single polycarbonate (PC) chains threaded into crystalline γ-cyclodextrin (γ-CD) channels were examined using solid-state ¹³C NMR and molecular dynamics simulations. The location of PC within the channels was confirmed by spin diffusion from a PC ¹³C label to natural-abundance ¹³C of the γ-CD. Rotor-encoded longitudinal magnetization (RELM) (under 7-kHz magic-angle sample-spinning conditions) was combined with multiple-pulse ¹H-¹H dipolar decoupling to detect large-amplitude phenyl-ring motion in both bulk PC and polycarbonate γ-cyclodextrin inclusion compound (PC-γ-CD). The RELM results indicate that the phenyl rings in PC-γ-CD undergo 180° flips faster than 10 kHz just as in bulk PC. The molecular dynamics simulations show that the frequency of the phenyl-ring flips depends on the cooperative motions of PC atoms and neighboring atoms of the γ-CD channel. The distribution of protonated aromatic-carbon laboratory and rotating-frame ¹³C spin-lattice relaxation rates for bulk PC and PC-γ-CD are similar but not identical. The distributions for both systems arise from site heterogeneities. For bulk PC, the heterogeneity is attributed to variations in local chain packing, and for PC-γ-CD the heterogeneity arises from variations in the location of the PC phenyl rings in the γ-CD channel. © 2007 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 45: 1271–1282, 2007

Local order in polycarbonate glasses by 13C{19F} rotational-echo double-resonance NMR

Co-reporter:Dirk Stueber;Anil K. Mehta;Zhiyun Chen;Karen L. Wooley

Journal of Polymer Science Part B: Polymer Physics 2006 Volume 44(Issue 19) pp:2760-2775

Publication Date(Web):18 AUG 2006

DOI:10.1002/polb.20931

Nearest-neighbor chain packing in a homogeneous blend of carbonate ¹³C-labeled bisphenol A polycarbonate and CF₃-labeled bisphenol A polycarbonate has been characterized using a shifted-pulse version of magic-angle spinning ¹³C{¹⁹F} rotational-echo double-resonance (REDOR) NMR. Complementary NMR experiments have also been performed on a polycarbonate homopolymer containing the same ¹³C and ¹⁹F labels. In the blend, the ¹³C observed spin was at high concentration, and the ¹⁹F dephasing or probe spin was at low concentration. In this situation, an analysis in terms of a distribution of isolated heteronuclear pairs of spins is valid. A comparison of the results for the blend and homopolymer defines the NMR conditions under which higher concentrations of probe labels can be used and a simple analysis of the REDOR results is still valid. The nearest neighbors of a CF₃ on one chain generally include a carbonate group on an adjacent chain. A direct interpretation of the REDOR total dephasing for the polycarbonate blend indicates that at least 75% of carbonate-carbon ¹³C···F₃ nearest neighbors are separated by a narrow distribution of distances 4.7 ± 0.3 Å. In addition, analysis of the variations in REDOR spinning-sideband dephasing shows that most of the ¹³C···F₃ dipolar vectors have a preferred orientation relative to the polycarbonate mainchain axis. This combination of distance and orientational constraints is interpreted in terms of local order in the packing of the carbonate group of one polycarbonate chain relative to the isopropylidene moiety in a neighboring chain. © 2006 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 44: 2760–2775, 2006

Vancomycin and Oritavancin Have Different Modes of Action in Enterococcus faecium

Co-reporter:Gary J. Patti, Sung Joon Kim, Tsyr-Yan Yu, Evelyne Dietrich, ... Jacob Schaefer

Journal of Molecular Biology (9 October 2009) Volume 392(Issue 5) pp:1178-1191

Publication Date(Web):9 October 2009

DOI:10.1016/j.jmb.2009.06.064

The increasing frequency of Enterococcus faecium isolates with multidrug resistance is a serious clinical problem given the severely limited number of therapeutic options available to treat these infections. Oritavancin is a promising new alternative in clinical development that has potent antimicrobial activity against both staphylococcal and enterococcal vancomycin-resistant pathogens. Using solid-state NMR to detect changes in the cell-wall structure and peptidoglycan precursors of whole cells after antibiotic-induced stress, we report that vancomycin and oritavancin have different modes of action in E. faecium. Our results show the accumulation of peptidoglycan precursors after vancomycin treatment, consistent with transglycosylase inhibition, but no measurable difference in cross-linking. In contrast, after oritavancin exposure, we did not observe the accumulation of peptidoglycan precursors. Instead, the number of cross-links is significantly reduced, showing that oritavancin primarily inhibits transpeptidation. We propose that the activity of oritavancin is the result of a secondary binding interaction with the E. faecium peptidoglycan. The hypothesis is supported by results from 13C{19F} rotational-echo double-resonance (REDOR) experiments on whole cells enriched with l-[1-13C]lysine and complexed with desleucyl [19F]oritavancin. These experiments establish that an oritavancin derivative with a damaged d-Ala–d-Ala binding pocket still binds to E. faecium peptidoglycan. The 13C{19F} REDOR dephasing maximum indicates that the secondary binding site of oritavancin is specific to nascent and template peptidoglycan. We conclude that the inhibition of transpeptidation by oritavancin in E. faecium is the result of the large number of secondary binding sites relative to the number of primary binding sites.

REDOR NMR Characterization of DNA Packaging in Bacteriophage T4

Co-reporter:Tsyr-Yan Yu, Jacob Schaefer

Journal of Molecular Biology (17 October 2008) Volume 382(Issue 4) pp:1031-1042

Publication Date(Web):17 October 2008

DOI:10.1016/j.jmb.2008.07.077

Bacteriophage T4 is a large-tailed Escherichia coli virus whose capsid is 120 × 86 nm. ATP-driven DNA packaging of the T4 capsid results in the loading of a 171-kb genome in less than 5 min during viral infection. We have isolated 50-mg quantities of uniform 15N- and [ε-15N]lysine-labeled bacteriophage T4. We have also introduced 15NH4+ into filled, unlabeled capsids from synthetic medium by exchange. We have examined lyo- and cryoprotected lyophilized T4 using 15N{31P} and 31P{15N} rotational-echo double resonance. The results of these experiments have shown that (i) packaged DNA is in an unperturbed duplex B-form conformation; (ii) the DNA phosphate negative charge is balanced by lysyl amines (3.2%), polyamines (5.8%), and monovalent cations (40%); and (iii) 11% of lysyl amines, 40% of –NH2 groups of polyamines, and 80% of monovalent cations within the lyophilized T4 capsid are involved in the DNA charge balance. The NMR evidence suggests that DNA enters the T4 capsid in a charge-unbalanced state. We propose that electrostatic interactions may provide free energy to supplement the nanomotor-driven T4 DNA packaging.

Oritavancin Exhibits Dual Mode of Action to Inhibit Cell-Wall Biosynthesis in Staphylococcus aureus

Co-reporter:Sung Joon Kim, Lynette Cegelski, Dirk Stueber, Manmilan Singh, ... Jacob Schaefer

Journal of Molecular Biology (14 March 2008) Volume 377(Issue 1) pp:281-293

Publication Date(Web):14 March 2008

DOI:10.1016/j.jmb.2008.01.031

Solid-state NMR measurements performed on intact whole cells of Staphylococcus aureus labeled selectively in vivo have established that des-N-methylleucyl oritavancin (which has antimicrobial activity) binds to the cell-wall peptidoglycan, even though removal of the terminal N-methylleucyl residue destroys the d-Ala-d-Ala binding pocket. By contrast, the des-N-methylleucyl form of vancomycin (which has no antimicrobial activity) does not bind to the cell wall. Solid-state NMR has also determined that oritavancin and vancomycin are comparable inhibitors of transglycosylation, but that oritavancin is a more potent inhibitor of transpeptidation. This combination of effects on cell-wall binding and biosynthesis is interpreted in terms of a recent proposal that oritavancin-like glycopeptides have two cell-wall binding sites: the well-known peptidoglycan d-Ala-d-Ala pentapeptide stem terminus and the pentaglycyl bridging segment. The resulting dual mode of action provides a structural framework for coordinated cell-wall assembly that accounts for the enhanced potency of oritavancin and oritavancin-like analogues against vancomycin-resistant organisms.

Oritavancin Binds to Isolated Protoplast Membranes but not Intact Protoplasts of Staphylococcus aureus

Co-reporter:Sung Joon Kim, Manmilan Singh, Jacob Schaefer

Journal of Molecular Biology (14 August 2009) Volume 391(Issue 2) pp:414-425

Publication Date(Web):14 August 2009

DOI:10.1016/j.jmb.2009.06.033

Solid-state NMR has been used to examine the binding of N′-4-[(4-fluorophenyl)benzyl)]chloroeremomycin, a fluorinated analogue of oritavancin, to isolated protoplast membranes and whole-cell sucrose-stabilized protoplasts of Staphylococcus aureus, grown in media containing [1-13C]glycine and l-[ɛ-15N]lysine. Rotational-echo double-resonance NMR was used to characterize the binding by estimating internuclear distances from 19F of oritavancin to 13C and 15N labels of the membrane-associated peptidoglycan and to the 31P of the phospholipid bilayer of the membrane. In isolated protoplast membranes, both with and without 1 M sucrose added to the buffer, the nascent peptidoglycan was extended away from the membrane surface and the oritavancin hydrophobic side chain was buried deep in the exposed lipid bilayer. However, there was no N′-4-[(4-fluorophenyl)benzyl)]chloroeremomycin binding to intact sucrose-stabilized protoplasts, even though the drug bound normally to the cell walls of whole cells of S. aureus in the presence of 1 M sucrose. As shown by the proximity of peptidoglycan-bridge 13C labels to phosphate 31P, the nascent peptidoglycan of the intact protoplasts was confined to the membrane surface.